Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Alberta, Canada.
Gastrointestinal Research Group and Inflammation Research Network, Department of Physiology and Pharmacology, Calvin, Joan and Phoebe Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Alberta, Canada.
Sci Rep. 2022 May 6;12(1):7453. doi: 10.1038/s41598-022-11123-w.
Intestinal homeostasis is highly dependent on optimal epithelial barrier function and permeability. Intestinal epithelial cells (IEC) regulate these properties acting as cellular gatekeepers by selectively absorbing nutrients and controlling the passage of luminal bacteria. These functions are energy demanding processes that are presumably met through mitochondrial-based processes. Routine methods for examining IEC mitochondrial function remain sparse, hence, our objective is to present standardized methods for quantifying mitochondrial energetics in an immortalized IEC line. Employing the murine IEC cell line, we present adapted methods and protocols to examine mitochondrial function using two well-known platforms: the Seahorse Extracellular Flux Analyzer and Oxygraph-2 k. To demonstrate the applicability of these protocols and instruments, IEC were treated with and without the murine colitogenic agent, dextran sulfate sodium (DSS, 2% w/v). Profound impairments with DSS treatment were found with both platforms, however, the Oxygraph-2 k allowed greater resolution of affected pathways including short-chain fatty acid metabolism. Mitochondrial functional analysis is a novel tool to explore the relationship between IEC energetics and functional consequences within the contexts of health and disease. The outlined methods offer an introductory starting point for such assessment and provide the investigator with insights into platform-specific capabilities.
肠道内环境的稳定高度依赖于上皮屏障功能和通透性的最佳状态。肠道上皮细胞（IEC）通过选择性吸收营养物质和控制腔内容物细菌的通过来调节这些特性，充当着细胞守门员的作用。这些功能是需要能量的过程，据推测是通过基于线粒体的过程来满足的。目前用于检查 IEC 线粒体功能的常规方法仍然很少，因此，我们的目标是提出一种标准化的方法，用于量化永生化 IEC 系中的线粒体能量。我们采用了鼠源 IEC 细胞系，提出了适应的方法和方案，使用两种著名的平台来检查线粒体功能： Seahorse 细胞外通量分析仪和 Oxygraph-2k。为了证明这些方案和仪器的适用性，我们用和不用鼠源结肠炎试剂，葡聚糖硫酸钠（DSS，2% w/v）处理了 IEC。在这两种平台上，DSS 处理都发现了明显的功能障碍，然而，Oxygraph-2k 允许更深入地了解受影响的途径，包括短链脂肪酸代谢。线粒体功能分析是一种探索 IEC 能量代谢与健康和疾病背景下功能后果之间关系的新工具。所提出的方法为这种评估提供了一个入门的起点，并为研究人员提供了对特定平台能力的深入了解。
